axor12: a novel human g protein-coupled receptor ... a novel human g protein-coupled receptor, named
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AXOR12: a novel human G protein-coupled receptor, activated by the peptide KiSS-1.
Alison I. Muir2, Larissa Chamberlain8, Nabil A. Elshourbagy6, David Michalovich5§, Darren J.
Moore8, Amy Calamari6, Philip G. Szekeres2, Henry M. Sarau7, Jon K. Chambers2, Paul
Murdock3, Klaudia Steplewski6, Usman Shabon6, Jane E. Miller2, Susan E. Middleton2, John G.
Darker4, Christopher G. C. Larminie5, Shelagh Wilson2, Derk J. Bergsma6 Piers Emson8, Richard
Faull9, Karen L. Philpott1, David C. Harrison1*
Depts of Neurology1, Discovery Biology2, Biotechnology and Genetics3, Discovery Chemistry4,
Bioinformatics5, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, CM19 5AW,
Depts of Biotechnology and Genetics6, Pulmonary Biology7 GlaxoSmithKline, 709 Swedeland
Road, King of Prussia, PA, USA
Neurobiology Programme, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK8
Dept of Anatomy with Radiology, University of Auckland, New Zealand9
§Current Address: Inpharmatica Ltd., 60 Charlotte St, London, W1T 2NU, UK
Telephone: +44 (0)1279 622728, Fax: +44 (0)1279 622371
Running Title: AXOR12, a novel receptor activated by KiSS-1
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on May 31, 2001 as Manuscript M102743200 by guest on A
pril 16, 2020 http://w
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A novel human G protein-coupled receptor, named AXOR12, exhibiting 81% homology to the
rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous
expression of AXOR12 in mammalian cells permitted the identification of three surrogate
agonist peptides, all with a common C-terminal amidated motif. High potency agonism,
indicative of a cognate ligand, was evident from peptides derived from KiSS-1, a gene whose
expression prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase
chain reaction (RT-PCR) was used to study the expression of AXOR12 and KiSS-1 in a variety
of tissues. Highest levels of expression of AXOR12 mRNA were observed in brain, pituitary
gland and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and
brain. A polyclonal antibody raised to the C-terminus of AXOR12 was generated and used to
show localization of the receptor to neurons in the cerebellum, cerebral cortex and brainstem.
The biological significance of these expression patterns and the nature of the putative cognate
ligand for AXOR12 are discussed.
by guest on A pril 16, 2020
The G protein-coupled receptors (GPCRs) form a large family of membrane bound proteins
which share a unique structural feature comprising 7 transmembrane alpha helices. These
molecules act as receptors for a diverse range of extracellular signalling molecules including
small molecules (amino acids, biogenic amines), lipids, small bioactive peptides and large
polypeptides (1). They have been successfully used as drug targets by the pharmaceutical
industry for a number of years. Attention has focussed on a number of proteins which are known
to be GPCRs through structural homology, but for which no ligand has been identified – so
called orphan receptors. At the same time as the recent discovery of new GPCRs, there has been
a renewed focus on discovering potential novel peptides which may act as endogenous ligands
for these receptors.
Here, we describe the cloning of a novel human orphan receptor, a class I GPCR with sequence
similarity to receptors for the neuropeptide galanin. This receptor was given the name AXOR12,
in accordance with its position in a series of receptors identified in our organization. AXOR12
has a high degree of homology to the rat orphan receptor GPR54 (2) (81% amino acid identity)
which suggests that these two receptors may be orthologs. In order to identify a ligand for
AXOR12 we expressed this receptor in mammalian cells, and screened the transfected cells in a
functional assay against a library rich in known and putative peptide transmitters. While there
was no activity in response to galanin, we identified three peptides which acted as low potency
agonists of AXOR12. These peptides were all derived from invertebrates and shared a C-
terminal LRF- or LRW-amide motif.
During the preparation of this manuscript, a search of patent literature revealed the existence of
additional, high potency agonists with sequence similarities to the surrogate agonists identified
by guest on A pril 16, 2020
from the screen. These peptides were derived from a precursor known as KiSS-1. The KiSS-1
gene was originally identified as being upregulated in melanoma cells which have lost the
potential to metastasize following microcell mediated transfer of human chromosome 6 (3).
Subsequent studies have shown that exogenous expression of KiSS-1 in other tumor cell lines
also prevents metastasis (4). The mechanism by which this occurs remains largely unknown,
however KiSS-1 has structural features that suggest that it may be the precursor of a secreted
peptide with an LRF-amide motif at the C-terminus. We synthesized the putative processed
products of KiSS-1 and observed that they acted as high potency agonists of AXOR12.
In order to gain insight into the physiological role of this receptor, we used quantitative RT-PCR
to localize the mRNA expression of AXOR12 and of KiSS-1 in a range of human tissues. We
observed high levels of AXOR12 expression in the brain. Further RT-PCR analysis of brain
expression revealed a distinctive pattern of mRNA localisation which was further explored by
immunohistochemistry using an antibody raised to the extreme C-terminal tail of the receptor. by guest on A pril 16, 2020
The human AXOR12 gene was initially identified within a genomic clone (AC005379) as five
coding exons interrupted by 4 introns. The full-length cDNA was obtained by a modification of
a previously described cDNA capture method (5). Briefly, 5µg of plasmid DNA from a human
brain cDNA library was screened with a biotinylated oligonucleotide (5'-biotin with 18 atom
spacer) corresponding to the 5' coding region (5'-GATGCGGACCGTGACCAACTTCTAC-3').
Two additional 40-mers ( 5'-GGAACTCGCTGGTCATCTACGTCATCTGCCGCCACAAGCC-
3' and 5'-ATCGCCAACCTGGCGGCCACGGACGTGACCTTCCTCCTGTG-3'),
corresponding to the immediate 5' and 3' regions of the biotinylated probe were also used as
blocking oligos. Bacterial colonies from the second round of selection were screened by PCR
using AXOR12 specific primers. Five positive clones were identified and the entire inserts were
sequenced on both strands using an ABI sequencer. Two of the sequenced clones were identical
to each other and to the full length AXOR12 cDNA predicted from the genomic DNA sequence.
Heterologous Receptor Expression and Functional Analysis in Mammalian Cells
The AXOR12 cDNA was subcloned into the mammalian expression vector pCDN (6) as
described previously (7). Transient transfections of HEK293 cells with AXOR12 alone or
AXOR12 co-transfected with Gqi5 (a chimeric G protein α subunit consisting of Gαq with the C-
terminal 5 amino acids substituted with the corresponding amino acids from Gαi2, which
facilitates GPCR signalling through phospholipase C) were prepared for functional studies. A
Ca2+ mobilization assay using the microtiter plate-based Ca2+ mobilization fluorometric imaging
plate reader (FLIPR) was performed as described previously (8). HEK293 cells co-transfected
by guest on A pril 16, 2020
with AXOR12 and Gqi5 were screened against a large library of over 1500 known and putative
GPCR agonists, including all available mammalian neuropeptides, as described previously (9).
Peptides in this library were tested at a final concentration of > 100 nM and other potential small
molecule agonists at > 1 µM.
To obtain mammalian cells stably expressing AXOR12, the cDNA was subcloned into the vector
pCD (a derivative of pCDN lacking the gene for neomycin resistance) and co-transfected with
pCDN Gqi5 (10) into Chinese hamster ovary cells using Lipofectamine PLUS (Life
Technologies) at a DNA ratio of 10:1 (CHO AXOR12:Gqi5 cells). Forty eight hours later the
cells were seeded into 96 well plates at 103 cells per well and selected with G418 (Life
Technologies) (400 µg/ml) and in the absence of nucleosides. Doubly selected cells were
screened by Northern blotting to confirm AXOR12 expression and positive clones we